The present invention, in some embodiments thereof, relates to a method for detecting non-transferrin bound iron.
The association between iron overload diseases and the occurrence of iron forms in plasma that are not associated with transferrin (Tf) bound iron (TBI) is a well-documented phenomenon [1,2]. These forms, referred collectively as non-TBI or NTBI, appear in plasma when Tf binding capacity for iron is exceeded due to massive iron overload, such as occurs in thalassemia major [3] and other congenital anemias [4], which require repeated blood transfusions, or bone marrow failure where iron utilization is inefficient [5]. NTBI may also occur in primary iron overload such as thalassemia intermedia or hemochromatosis even in the setting of <100% Tf saturation [6,7,8], due apparently to formation of insoluble polynuclear ferrihydrate species [9] and complexes with modified albumin [10] that are inaccessible to Tf.
The initial study of NTBI in the serum of thalassemia patients [11] and others that followed [reviewed in 12] led to the recognition that NTBI may be an important indicator of systemic iron overload and source of tissue iron accumulation. However, studies have highlighted difficulties in analyzing NTBI in a complex medium, which necessitated usage of chelating or mobilizing agents to solubilize it and facilitate its separation from TBI by ultrafiltration, prior to chemical detection. NTBI appears in iron overloaded plasma in multiple forms, some easily filterable via size exclusion leaving others protein bound, some complexed to small organic ligands which, in turn, might be free or adsorbed to proteins [9,10]. Moreover, the composition of NTBI might vary with the degree and source of iron overload, treatment of the patient with chelator or phlebotomy and the sample storage conditions which, ideally, should preserve both the TBI and the NTBI components. Due to the chemical complexity and potential clinical importance of NTBI, a number of alternative methods have been developed for its detection [12,13] with the objective of obtaining a better measure of the total content of NTBI in a patient sample. In general these methods have relied on the principle of using a powerful chelator or high concentrations of mobilizing agents (with or without a filtration step), in order to maximize NTBI' s accessibility to the various iron-detecting reagents [11,14-16].
The present inventors have previously developed an assay for quantifying the overtly labile forms of NTBI in plasma or serum, namely endogenous forms that are both redox-active, exchangeable and chelatable, which they defined as labile plasma iron or LPI [8,17]. The chemical properties of LPI in native plasma were assumed to be pathophysiologically relevant as a source of tissue iron overload because 1. ligand exchangeability is a precondition for metal transfer across membranes via transporters/ channels and 2. LPI can serve as direct chelator target [1]. The advantage of the LPI assays is their performance on native plasma or serum, which essentially avoids potential complications associated with the use of high concentrations of iron-mobilizing agents or filtration steps by other NTBI assays [14,15]. The disadvantage of using just native plasma relates to variable plasma components (e.g. citrate, uric acid and albumin) that have the potential to dampen LPI measurements.